In this work, spherical core-shell-shell particles based on a gold core, a fluorescently labelled silica shell and a non-fluorescent silica shell were self-assembled into colloidal crystal to form regular 3D lattices. This sample was used as a ruler to calibrate a confocal microscope and to align the beam of a ... read more 3D-stimulated emission
depletion (STED) microscope.
First, 15 nm diameter gold nanospheres were used as a metal core. However, the reflectance signal of these cores was low. This was observed when the refractive index (RI) of the embedding mixture was matched with the RI of the silica shell, which was measured in this study (n = 1.4448). Consequently, particles consisted of a 30 nm diameter gold core, a 80 nm diameter fluorescence core, with Rhodamine B isothiocyanate (RITC) as the fluorophore, and a total diameter of 500 nm (&lt;5% PD). Thus, the particles self-assembled into a colloidal crystal via a vertical deposition with a small angle inside an oven at 50 °C.
The distances between particles in the colloidal crystal were measured with the confocal and compared with the effective distance determined via the position of the band gap in the Fourier transform infrared (FTIR) spectrum. A calibration error of 3.93%, 10.14%, and 7.08%, in the x-,y-, and z- direction respectively, was determined. The gold cores were used to observe and align the overlapping of the excitation and depletion beams of the STED microscope.
A better localisation of the band gap and more distance averaging will result in a more reliable calibration. Apart from this, it was observed that not all the particles should present a metal core to allow alignment of the excitation and depletion beams. Hence, only ~1% of the particles should have a gold core. Furthermore, coreshell-sell particles with 5(6)-carboxyfluorescein (CF) as the fluorophore were also synthesised. A binary colloidal crystal containing both kind of dyed particles was fabricated. However, further optimization on the dye labelling of CF is required. Nonetheless, the promising results of this work show the potential fabrication of this bi- or trinary colloidal crystals and its usage as a ruler to calibrate in three dimensions more than one STED laser with one sample. show less

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